A familial Alzheimer’s disease associated mutation in presenilin-1 mediates amyloid-beta independent cell specific neurodegeneration

Mutations in the presenilin (PS) genes are a predominant cause of familial Alzheimer’s disease (fAD). An ortholog of PS in the genetic model organism Caenorhabditis elegans (C. elegans) is sel-12. Mutations in the presenilin genes are commonly thought to lead to fAD by upregulating the expression of amyloid beta (Aβ), however this hypothesis has been challenged by recent evidence. As C. elegans lack amyloid beta (Aβ), the goal of this work was to examine Aβ-independent effects of mutations in sel-12 and PS1/PS2 on behaviour and sensory neuron morphology across the lifespan in a C. elegans model. Olfactory chemotaxis experiments were conducted on sel-12(ok2078) loss-of-function mutant worms. Adult sel-12 mutant worms showed significantly lower levels of chemotaxis to odorants compared to wild-type worms throughout their lifespan, and this deficit increased with age. The chemotaxis phenotype in sel-12 mutant worms is rescued by transgenic over-expression of human wild-type PS1, but not the classic fAD-associated variant PS1C410Y, when expression was driven by either the endogenous sel-12 promoter (Psel-12), a pan-neuronal promoter (Primb-1), or by a promoter whose primary expression was in the sensory neurons responsible for the chemotaxis behavior (Psra-6, Podr-10). The behavioural phenotype was also rescued by over-expressing an atypical fAD-linked mutation in PS1 (PS1ΔS169) that has been reported to leave the Notch pathway intact. An examination of the morphology of polymodal nociceptive (ASH) neurons responsible for the chemotaxis behavior also showed increased neurodegeneration over time in sel-12 mutant worms that could be rescued by the same transgenes that rescued the behaviour, demonstrating a parallel with the observed behavioral deficits. Thus, we report an Aβ-independent neurodegeneration in C. elegans that was rescued by cell specific over-expression of wild-type human presenilin.


Introduction
There are a variety of disorders that lead to impaired memory and cognition among older adults, of which Alzheimer's disease (AD) is the most common, accounting for 60% of all dementias [1].The majority of AD and related dementias are chronic illnesses; they appear in stages, and patients' symptoms worsen with time [2].There are two forms of AD: sporadic AD where genetic risk factors are inherited in a non-Mendelian fashion, and familial AD (fAD) where genetic risk factors are autosomal dominant [3].Familial AD presents earlier than the sixth or seventh life decade and is much less prevalent than AD, however, because fAD offers known genetic causes, this form of the disorder has been the target of much research.Mutations in three genes have been linked to a majority of fAD cases: presenilin 1 (PS1), and presenilin 2 (PS2) and amyloid precursor protein (APP; [4]).Although these genes each alter a large number of cellular processes, multiple studies have focussed on a two common effects: mutations in any one of these genes lead to increased production of amyloid-beta (Aβ) and inflammation [5].
The presenilins have a number of intracellular functions; the most commonly studied is their role as the catalytic subunit of γ-secretase, a protein responsible for the proteolysis of many proteins including APP and Notch [6].To date, more than 350 PS1 mutations have been reported in AD patients [7].A second protein, PS2, has significant homology to PS1 at both gene and protein levels and has also been identified as a cause of fAD [8].Mutations in PS2 account for the smallest percentage of fAD cases and lead to a later age of onset compared to PS1 and APP mutations [4].In the clinic, the majority of presenilin mutations identified in patients are missense substitutions that present an autosomal dominant inheritance pattern.As dominant phenotypes typically imply a gain-of-function mechanism, it is believed that pathogenic mutations in PS lead to a toxic gain-of-function phenotype that increases Aß42/ Aß40 ratios, a well-known biomarker of AD [9].However, more recent analysis using mammalian cells has shown that while a vast majority of the studied PS1 mutations do lead to increased Aß42/Aß40 ratios, a majority of these variants significantly decrease production of both Aß42 and Aß40 and have no significant correlation to mean age at onset of fAD for the corresponding mutations [10], casting doubt on the widely accepted assumption that pathogenic PS1 variants lead to fAD by favoring production of Aß42 over Aß40.
In studies of the underlying causes of AD, APP has been a major focus of research.The amyloid hypothesis was formalized for the first time in 1992 when Hardy and Higgins proposed that the build-up of plaques containing Aβ is the predominant cause of AD [11].Since then, many studies have attempted to determine how amyloid plaques lead to AD and whether elimination of plaques would cure AD.Thus far, research on amyloid plaques has not led to clear answers or to an effective treatment for AD.Although amyloid plaques are considered a hallmark of AD, a build-up of amyloid peptides can also be part of normal aging as Aβ plaques are apparent in 45% of cognitively normal elderly individuals [12].Because the strongest predictor of both Aβ build up and AD is aging, it may be that other aging-related issues combine with the build-up of amyloid plaques to trigger AD.Since the aggregation of Aβ plaques cannot, on its own, be the sole causal mechanism underlying AD, researchers are exploring the notion that AD is a complex disorder and that multiple pathways other than amyloid plaques may produce the disease characteristics [13].
Because of the known role of presenilins in processing APP, much of the research done on PS1/PS2 is within the context of processing APP.Mouse models containing only PS1 or PS2 mutations show an increased proportion of Aβ 42 but do not exhibit amyloid plaques, thus numerous rodent studies investigating PS1 have used double transgenic mice that have mutations in both APP and PS1 transgenes [14,15].Plaque formation and cognitive impairment phenotypes manifest much earlier and much more extensively in these double transgenic lines, compared to APP transgenic lines without PS1/PS2 mutations or transgenes [14].
Somewhat less studied in rodents are phenotypes in transgenic lines expressing only presenilin mutations, possibly due to lack of complete AD-like pathology.However, recent rodent work has shown that PS1 and PS2 fAD transgenic lines show a plethora of phenotypes, including increased susceptibility to hippocampal damage from kainite induced excitotoxicity or trimethyltin treatment, increased protein oxidation and lipid peroxidation in the mutant brain, impaired hippocampal neurogenesis in adult mice and an age-dependent impairment of spine morphology and synaptic plasticity in hippocampal neurons [15,16].Partial or complete loss of presenilin by the knock-out of both PS1 and PS2 leads to age-dependent neurodegeneration in the mouse brain [17][18][19], and in another PS1 knock-in line aging-dependent neurodegeneration and neuronal loss has also been observed [20].In electrophysiological studies, PS1 fAD mutations were found to alter long-term potentiation (LTP) in the hippocampus [16].In studies comparing very young transgenic mice over-expressing an fAD mutation in PS1 to counterparts over-expressing wild-type PS1, fAD mutants showed enhanced LTP.However, at 8-10 months of age LTP was similar to wild-type mice, and at 13-14 months of age LTP was significantly decreased [16].Pharmacological blocking studies also showed that the fAD-associated PS1 mutants altered NMDA receptor-mediated neurotransmission [21].Behavioural experiments in young mice have also shown that PS1 fAD transgenic mice have behavioural impairments, although the deficits observed are somewhat subtle and inconsistent [16].Taken together, there is evidence to suggest an alternate hypothesis that loss-of-function mutations in the presenilin genes can elicit AD pathology through one or more molecular pathway(s) independent from that for processing APP [22].
Another proteolytic target for γ-secretase is Notch.Alterations in Notch proteolysis by mutations in PS1 that affect γ-secretase may also be involved in AD pathogenesis.The Notch protein is a type I transmembrane cell surface receptor that plays a role in cell fate decisions of both vertebrates and invertebrates [23,24].Binding to a member of the Delta, Serrate, or Lag2 (DSL) family of ligands triggers Notch proteolysis, leading to the cleavage of Notch by many of the same secretases that cleave APP [25].Notch is expressed in neurons at especially high levels in the hippocampus in the adult rodent brain [26], and in some rodent models with presenilin deficiencies, Notch loss-of-function phenotypes are observed [27,28].Thus, there is evidence to suggest that alterations in Notch can be a contributing factor to AD pathogenesis in the context of PS1.To date the role of Notch signaling in AD pathogenesis remains largely unclear.
In the present study, we used the roundworm Caenorhabditis elegans (C.elegans) to investigate the role of PS1 and Notch in neural degeneration and relevant behavioral impairments produced by a mutation in sel-12, an orthologue of PS1.SEL-12 shares approximately 50% amino acid identity with human PS1, among which include many AD-associated residues.As C. elegans express two genes orthologous to human Notch receptors, glp-1 and lin-12, questions regarding Notch signaling in the context of fAD can be interrogated in this model system as well.There are a number of advantages to using C. elegans for studying neurological degenerative diseases including its short life span (14-18 days), a sequenced genome, and defined nervous system with 302 identified neurons that are well characterized with a mapped connectome [29].Basic cellular neurobiological functions are well conserved, with C. elegans having cellular and biochemical processes similar to those in mammals.The first known function of presenilins was the role of PS1 in cleaving Notch based on the observation that mutations in sel-12 produced an egg-laying deficit known to be caused by impaired Notch signaling [30].C. elegans shows both genetic and functional similarity with orthologs of many of the mammalian neurotransmitters (e.g., dopamine, serotonin, GABA, glutamate, acetylcholine, and their receptor subtypes) and many classes of ion channels (e.g., sodium, calcium, and potassium channels) [31].In fact, in addition to PS1, a number of human genes associated with AD have C. elegans orthologs [32].However, it is important to acknowledge that the C. elegans ortholog of APP, apl-1, does not generate a product orthologous to Aβ nor does the nematode system have an ortholog of BACE-1 encoding β-secretase [29].Whereas the human APP protein may undergo two primary proteolytic pathways (the non-amyloidogenic involving the α-and γsecretases, and the amyloidogenic pathway involving β-and γ-secretases), the orthologous nematode system has only the orthologous proteolytic components for the non-amyloidogenic pathway.However, besides lacking a β-secretase and an orthologous Aβ sequence, the APP processing pathway is largely conserved [33,34] with experimental evidence suggesting that SEL-12 regulates the activity of APL-1 [35].Therefore, the nematode model offers a unique opportunity for studying functions of presenilins in an Aβ-free biological system.
Here we studied whether mutations in sel-12 altered chemotaxis and sensory neuron morphology across aging, and whether deficits could be rescued by over-expressing variants of human PS1.To do this we first tested olfactory chemotaxis across development and during aging in sel-12 mutant worms, and in worms over-expressing either human wild-type PS1 or one of the two PS1 mutations: PS1 C410Y , a classical fAD mutation that impairs both Aβ and Notch processing, [36,37], and a novel mutation, PS1 ΔS169 , which is believed to impair Aβ but leave Notch processing intact [38].In sel-12 mutants and worms over-expressing the PS1 transgenes, we also assessed whether there was neurodegeneration of the ASH chemosensory neurons that are responsible for sensing the odorant used in our chemotaxis assay.We found that in sel-12 mutant worms, there was more degeneration of the ASH sensory neurons with increasing age and this degeneration could be rescued by both nervous system and cell-specific expression of wild-type PS1 and PS1 ΔS169 , but not PS1 C410Y .These results show an Aβ-independent, cell specific role of presenilin in neuronal degeneration.

Generation of transgenic lines and strain maintenance
C. elegans Bristol wild-type (N2), sel-12(ok2078) mutant (RB1672), and the transgenic nuIs [osm-10::GFP+lin-15(+)] (HA3) strains were provided by the Caenorhabditis Genetics Center (CGC), and the transgenic strain HA1712 (lin-12 (null); Phsp-16.2::glp-1RNAi) was provided graciously by Dr. Anne Hart of Brown University.Worms were cultured on Escherichia coli (E.coli) seeded nematode growth medium (NGM).All experiments were conducted in 6cm Petri plates that were filled with 10 mL NGM agar a maximum of two weeks prior to use.Worms were stored in a 20˚C incubator and all experiments were conducted in a room with controlled humidity (40±5% RH) and temperature (20±1˚C).
Transgenic overexpression lines were constructed by microinjection of DNA plasmids into the germ line of young adult sel-12 mutant worms at a concentration of 10 ng/μl along with a co-injection GFP marker [39].This DNA mixture was injected into the distal arm of the worm's gonad, which contains a central cytoplasm core that is shared by germ cell nuclei [39].Worms were injected under an inverted Zeiss DIC microscope equipped with a 40X Nomarski objective.The plasmid pBY140 containing the wild-type PS1 coding region expressed by the sel-12 promoter was provided by Dr. Ralf Baumeister (Albert-Ludwig University in Freiburg/ Breisgau, Germany).To generate pan-neuronal, ASH-specific and AW-specific over-expression constructs, promoter regions of rimb-1 (2.6kb fragment, 734bp upstream of start codon, from pSF11[ptag-168p::nCre]), sra-6 (1.8kb fragment, 158bp upstream of start codon) and odr-10 (986bp fragment, 28bp upstream of start codon), respectively were used to replace the sel-12 promoter region in the original pBY140 via restriction cloning.Lines with >50% GFP transmission were selected for testing.Because DNA microinjection creates an extra-chromosomal array, there is inherent variability of expression levels because there were probably different copy numbers of plasmids incorporated into the array and there was also the possibility of different cells having different genotypes (mosaic expression) [40].To overcome this, three independent lines for each construct were tested [41].Please refer to S1 Table for a full list of strains generated and tested.

Chemotaxis assays
To ensure that all animals tested were the same developmental age, adult worms (n = 20-30) were placed in a droplet of 4μl of bleach solution (1:1 ratio of 100% bleach and 1M NaCl).The worms' bodies disintegrated but their eggs survived.All eggs hatched at roughly the same time, and larvae were allowed to grow for three days on agar seeded with E. coli in a 20˚C incubator.On day three, 72-hour post egg-lay worms were collected using 700μl of liquid M9 buffer into 1.5mL centrifuge tubes.To remove OP50 E. coli from the bodies of the worms, worms were centrifuged for a minute at 1000rcf and the supernatant was removed.Another 700ul of M9 buffer was added to the tubes, and the worms were centrifuged again (done 3 times).After the third time, the supernatant was removed and the pellet of the worms remained in the tube.
The chemotaxis procedure used was adapted from Margie et al. [42].Eight NGM Petri plates (6cm diameter) were used per strain (4 control, 4 test) and each was divided into four equal quadrants.In the center of each plate a circle of 1cm diameter (Fig 1A) was drawn on the bottom of the plate and lines were drawn to mark the quadrants.For test plates, each quadrant was marked as control or test.For control plates, all 4 quadrants were labelled as control.The octanol solution was prepared by mixing of a 1:1 ratio of 100% octanol and 1M-sodium azide (NaN 3 ) that was used to immobilize worms.The diacetyl solution was prepared by diluting it to 0.5% diacetyl using 99.5μl of M9 and 5μl of diacetyl.A 1:1 ratio of this 0.5% diacetyl solution was then added to NaN 3 .Control solution was prepared by mixing equal volumes of M9 and NaN 3 .Washed worms (50-100) were pipetted into the circle at the center of the plates.Then 2μl of the volatile odorants octanol or diacetyl were placed on spots located at the centre of the test quadrants, and 2μl of the control solution was placed on spots located at the centre of the control quadrants (Fig 1A).
Plate lids were kept open for approximately 15 minutes until the odorants soaked into the agar and the surface of the agar appeared dry.Worms were then left to move around the plates undisturbed for one hour in a 20˚C room.Plates were then transferred into a 4˚C refrigerator for two hours to immobilize worms before counting.Prior to counting plates were relabeled and a key generated, plates were removed from the refrigerator for counting one at a time and the number of worms in each quadrant was counted by a researcher blind to the condition/ strain/the study objectives.After counting the blind was broken and a chemotaxis index (CI) was calculated (Fig 1A ).

Use of 5'-fluorodeoxyuridine (FUdR) in aging studies
Because sel-12 mutant worms have an egg-laying deficit, eggs accumulate inside the gonad and the progeny hatch inside the adult killing it at a relatively young age (Levitan et al., 1995).This makes studies using older worms difficult.To stop the hatching of eggs inside adults so the adults lived to an older age, we used 5'-fluorodeoxyuridine (FUdR).FUdR is an inhibitor of DNA synthesis that is used in C. elegans lifespan studies to preserve a synchronized aging population [43].Two days prior to adding E. coli to NGM plates, 250μl of 100μM FUdR was spread on plates.Two days after the addition of E. coli, Larval stage 4 (L4, approx.50 hours post hatch) animals were transferred to the NGM plates with FUdr.For aging studies, adult worms

Locomotion assay
Behavioral experiments on worms of different ages were used to determine speed and the locomotory ability of worms on plates to ensure they were able to chemotax to the test spots in the time given.The multi-worm tracker (MWT) is an automated machine vision system that records features of worm behavior for many worms simultaneously; for this assay crawling speed was measured [44].To assess locomotion, approximately 50 worms of each age were handpicked onto a plate without FUdR or E. coli (n = 3 plates), placed on the MWT, and the lid of each plate was lifted briefly to provide an air-puff stimulus to arouse worms so they were moving and could be recognized by the MWT.Worms were tracked for 250 seconds.

Knock-down of both Notch receptors via heat shock and developmental stage synchronization
Because worms carrying null mutations in both glp-1 and lin-12 have a lethal phenotype, we studied the effect of double loss-of-function of both Notch receptors by the adulthood induction of glp-1 RNAi knockdown using a heat-shock promoter (hsp-16.2) in a lin-12(n941) null mutant background.Animals with a heat shock promoter driving glp-1 RNAi were raised at 15˚C, the permissive temperature.During heat-shock experiments, all strains used, including wild-type, were moved to 33˚C, the restrictive temperature, for 2 hours as described in Singh et al. [45] After this, animals were allowed to recover for 3 hours in a 20˚C incubator prior to chemotaxis experiments that were conducted in a 20˚C behavior room.Expression levels of hsp-16.2persist even up to 50 hours after heat shock [46], suggesting that after 3 hours of recovery there would still be sufficient knockdown of glp-1.Note that Notch mutant worms have developmental delays compared to wild-type.To test worms at similar stages in development, wild-type and lin-12/glp-1 deficient worms were closely observed every 4 hours to determine when these strains reached young adulthood (defined as containing at least 4 eggs [47]).Compared to wild-type worms, worms with deficiencies in both Notch receptors had a 6-hour lag in development (reached young adults at 68 hours in 20˚C).

ASH neuron imaging
The single pair of ASH sensory neurons in the head of the worm are the primary chemosensory receptors for detecting octanol.For imaging we used strains expressing Posm-10::GFP that filled the ASH neuron cell bodies and processes with GFP and imaged both wild-type and sel-12 mutant worms over time (78, 98, and 108 hour old worms).For all extrachromosomal rescue strains, the line that had the closest chemotaxis index to wild-type was chosen for imaging.The researcher doing the imaging was blind to the strains being imaged.Worms were placed on 2% agar pads on sterile glass microscope slides in 15uL of 50mM NaN 3 for immobilization.Worms were given approximately one minute to become immobile prior to being covered with a 1.5mm thick coverslip.Images were obtained using a Leica SP8 white light

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confocal microscope.To excite GFP, a 488nm wavelength laser was used and the emitted light was collected by passing through a 510-550nm bandpass filter.Depending on the thickness of the worm and the brightness of its GFP, optical sections were collected at various intervals using a 63X oil immersion lens and summed into a single Z projected image.Neurons were quantified using the Fiji: ImageJ Software.
Independent groups of one hundred worms were imaged per strain at 78, 98 and 108 hours of age for a total of 300 images per strain.During analysis, ASH neuron morphology was classified into three categories: normal, gapped/missing arm, and bleb.If gaps and blebs were both present, the neuron would be categorized as blebs.
For DiI dye-filling experiments, animals were collected and incubated in 400μL 1.25μM DiI in the dark for 30 minutes.After incubation, worms were transferred back onto OP-50 seeded NGM plates for 30 minutes then collected for imaging.

Statistical analysis
Data are reported as means ± standard error of the mean (SEM) of three or four independent plates with 50-100 worms on each for each strain tested.Because expression levels from extrachromosomal transgenes can be variable, we analyzed data for 3 independent lines of each transgene.For the sake of clarity, we report analysis for our collapsed data.The presenilin rescue strains are reported as the average of four plates for each of three extrachromosomal lines (total of 12 plates).Each rescue line was analyzed independently and then as averages of the three extrachromosomal rescue lines for each strain.Data analyses were conducted using the GraphPad Prism software, SPSS and python (pingouin 0.5.1).Data were checked for normal distribution and one-tailed independent T tests were conducted for pair-wise comparisons.In experiments with three or more groups, a one-way ANOVA was conducted with a Tukey's Honest Significant Difference test with statistical significance set at p = 0.05 for post-hoc analyses.The percentage of normal and abnormal ASH morphology of each strain was compared to other strains using aggregated Pearson's chi-squared test.P values of less than 0.05 were considered significant.Data visualization was done with GraphPad Prism, Python (with Plotly 5.7.0 graphing library) and Adobe Illustrator.

C. elegans with a sel-12 mutation displayed olfactory impairments shortly after hatching that increased over time
In C. elegans, there are three members of the Presenilin family: hop-1, sel-12, and spe-4 [41,48,49].spe-4 is only distantly related to human presenilins and is expressed only in the male germline [48], and thus was not investigated in this work.Although both hop-1 and sel-12 are homologous in terms of protein structure and domain similarity to human PS1, are widely expressed throughout the worms [30] and have been shown to function redundantly in facilitating the Notch pathway through lin-12 and glp-1 signalling [49][50][51], hop-1 is considered more diverged from SEL-12 and PS1 (31% sequence homology to human PS1 compared to 50% for SEL-12).Additionally, of the 113 PS1 residues where pathogenic and likely pathogenic occur, only 45 (40%) are conserved in HOP-1 whereas 89 (79%) are conserved in SEL-12 (S1 Fig) .Critically, whereas sel-12 is strongly expressed in the neurons studied in our experiments (ASH and AWA), hop-1 is not [52].Thus, we focused our work on a strain of worms expressing a putatively null sel-12(ok2078) deletion allele.
To assess the olfactory abilities of wild-type and sel-12 mutant worms across the lifespan we examined them across development.C. elegans have 4 larval stages (lasting from ~12-24 hrs in length) and we tested the aversive chemotaxic response to octanol at each stage, L1-L4, as well as at 72 hours post egg-lay (reproductive young adult worms; Fig 1B).Compared to wild-type worms, sel-12 mutants had significant chemotaxis index deficits from L1 that were also seen in L2, L3, L4, and adult worms [F (9, 30) = 65.60,p <0.001] (Fig 1C).This finding was surprising, as past observations noted that chemotaxis towards food was not effected in sel-12 mutants [51].
We also tested whether the olfactory deficits progressed with advanced age by testing worms ranging in age from reproductive adult to old age at 10 hour intervals (78-108 hours post egg-lay; Fig 1D).The multivariate analysis of covariance showed a significant group effect indicating there was a significant difference in CI scores between groups ([F(7, 24) = 60.11,p<0.001]).Post-hoc analyses showed that although there was a mild but non-significant pattern of decrease in chemotaxis index over time between 78 hours old and 108s hour old in wild-type worms (p = 0.06), the decrease in chemotaxis index was much more rapid and was significant in sel-12 mutant worms across age (Fig 1D; p = 0.05).Overall, wild-type and sel-12 mutant worms had chemotaxis index decrements from 78 to 108-hours old of 15% and 81%, respectively.

Motor ability is not a confounding variable in sel-12(ok2078) mutant chemotaxis
It is possible that the egg-laying phenotype of the sel-12 mutants could lead to bloating and impair the ability to locomote well enough to successfully chemotax within the allotted time.We investigated this possibility by assessing locomotor speed of wild-type and sel-12 worms at 68hr, 78hr, 88hr, 98hr and 108hrs of age on the MWT, in a neutral E. coli (OP-50) seeded environment without any odourants.Although at 68 hours old sel-12 mutant worms appeared slower than wild-type by visual observation, via MWT analyses average forward speed during the entire tracking session was not significantly different (S2A Fig; p = 0.08).In comparing worms aged from 68 hours to 108 hours old, wild-type worms' forward speed peaked at 88 hours and slowed down at 108 hours old.In contrast, the speed of sel-12 mutant worms did not increase from 68 hours to 88 hours old and was significantly slower at 88-hour (t(4) = 5.89, p = 0.002) and 98-hour old (t(4) = 6.10, p = 0.002) compared to wild-type worms (S2A Fig) .Despite the observation that sel-12 mutant worms move more slowly than wild-type worms, they were able to navigate around the entire petri plate within approximately 5 minutes (S2B Fig), demonstrating that they could exhibit chemotaxis behavior in the 1-hour test session if they were able to detect the olfactory cue.Thus, despite a marked reduction in locomotion speed in comparison to their wild-type counterparts, sel-12 mutants maintained sufficient locomotor ability to explore and navigate to all areas of the plate environment in the given time.

Expression of wild-type sel-12 and wild-type PS1 rescued chemotaxis deficit when driven by the endogenous sel-12 promoter
Using extrachromosomal arrays we over-expressed wild-type sel-12 with its endogenous promoter (Psel-12::sel-12) in the sel-12 mutant and showed that it rescued octanol chemotaxis behavior (Fig 2A;  .001]).This finding was consistent with a previous study testing functional conservation between sel-12 and PS1 in which over-expressing human wild-type PS1 using the endogenous sel-12 promoter in a sel-12 mutant rescued mutant phenotypes [30].Because sel-12 is expressed in both neuronal and non-neuronal cells in the worm, we next tested whether the octanol chemotaxis deficit could be rescued by expressing PS1 in just the nervous system.
In sel-12 mutant animals an attractive chemotaxis response to diacetyl was also impaired.Using the same strains, we saw that over-expressing sel-12, PS1 WT and PS1 Δs169 pan-neuronally also rescued impaired diacetyl chemotaxis, while expressing PS1 C410Y did not (S4 Fig).

ASH neuron-specific expression of wild-type sel-12, wild-type PS1, and PS1
Δs169, but not PS1 C410Y, rescued chemotaxis deficits in sel-12 mutant worms Thus far, our results have aligned with previous studies that show that over-expression of human PS1 in a tissue-specific manner is able rescue phenotypes caused by sel-12 loss-of-function, suggesting cell-autonomous function [30,51,54].To take things further, we investigated whether sel-12 acts in a cell-specific manner through double-dissociation.In C. elegans, the ASH sensory neurons are polymodal nociceptors that detect a number of noxious and potentially toxic stimuli including octanol [55].The avoidance response to octanol is mediated by ASH activation [55].To determine whether presenilin plays a role in chemotaxis by acting specifically in these sensory neurons, we over-expressed sel-12, PS1 WT , PS1 C410Y , and PS1 Δs169 in Next, we over-expressed wild-type human PS1 under the control of the sra-6 promoter (Fig 3B ), and found that it rescued the chemotaxis impairments observed in sel-12 mutant worms ([F(2,17) = 44.29,p<0.001]; wild-type vs PS1 WT rescue p = 0.28, sel-12 mutant vs. PS1 rescue p<0.001).On the other hand, when we expressed the canonical fAD PS1 mutation C410Y in the ASH neurons (Psra-6::PS1 C410Y ; Fig 3C ), we found that it did not rescue chemotaxis deficits in sel-12 mutant worms ([F(2,17) = 16.68,p<0.001]; wild-type vs.PS1 C410Y rescue p<0.001, sel-12 mutant vs. PS1 C410Y rescue p = 0.72).Finally, in contrast to Psra-6: PS1 C410Y over-expression, Psra-6::PS1 Δs169 over-expression did restore chemotaxis deficits in sel-12 mutant worms (Fig 3D; [F(2,17) = 30.69,p<0.001]; wild-type vs.PS1 Δs169 rescue p = 0.92, sel-12 mutant vs. PS1 Δs169 rescue p<0.001).This data aligns with previous studies that show that there is functional conservation between human PS1 and its nematode ortholog.The loss-offunction mutations of both PS1 and its nematode ortholog appear to impair cell function in a cell autonomous manner as over-expressing these genes in a single pair of sensory neurons rescued the sel-12 chemotaxis deficit.
Wild-type PS1 rescue lines in the ASH neuron had a cell-specific effect on chemotaxis.Although wild-type PS1 in ASH was sufficient to rescue octanol chemotaxis, it is possible that the rescue was not cell-autonomous, and that expression of PS1 WT anywhere in the nervous system would rescue the phenotype.To rule out this hypothesis, we expressed wildtype PS1 driven by the odr-10 promoter that is expressed in another pair of chemosensory neurons, the AWA neurons, which are responsible for chemotaxis towards the attractant diacetyl.Consistent with our ASH-octanol avoidance findings, worms with the same mutation in sel-12 are also deficient in appetitive chemotaxis towards diacetyl and they show the same pattern of chemotaxis deficits and rescues for PS1 WT and PS1 mutations as was observed for aversive chemotaxis away from octanol (S5A-S5C Fig).
We then tested whether AWA-specific over-expression would rescue ASH-specific phenotypes, and whether ASH-specific over-expression would rescue AWA-specific phenotypes.Over-expression of wild-type PS1 in the AWA neurons did not rescue chemotaxis impairment in octanol avoidance ( Thus, expression of PS1 WT in the ASH neurons rescued chemotaxis deficits in sel-12 mutant worms for the octanol odorant, but not for the diacetyl odorant sensed by different sensory neurons, the AWA neurons.At the same time expression of PS1 WT in the AWA sensory neurons did not rescue octanol chemotaxis but did rescue chemotaxis to diacetyl.Therefore, via double-dissociation we show that the roles of sel-12 in mediating the chemotaxis deficits are cell specific, and cell-autonomous.

C. elegans with mutations in Notch receptors do not show increased chemotaxis deficits over time
Contrasting chemotaxis data from animals with the two fAD mutations, the classic mutation PS1 C410Y that alters both Aβ and Notch cleavage, and PS1 Δs169 mutation that affects Aβ cleavage but leaves Notch intact, suggested an important role for Notch cleavage by sel-12 in normal chemotaxis.Like APP, Notch receptors are cleaved and activated by the presenilins of γ-secretase.In earlier research it was reported that in C. elegans the two Notch receptors, lin-12 and glp-1, redundantly regulate chemosensory avoidance behavior in response to octanol [57].As single mutations of either glp-1 or lin-12 did not show chemotaxis impairments [57], we used the transgenic strain created by Singh et al. in which glp-1 was knocked down using heat-shock promotor driven RNAi in adult lin-12(n941) mutants, and tested octanol chemotaxis of this strain as worms aged to determine whether these deficits increased in the same way as did chemotaxis in the sel-12 mutant worms.
Both sel-12 mutant worms and lin-12/glp-1 mutant worms had significantly lower CIs compared to wild-type worms (p <0.001; Fig 5).Compared to age-matched wild-type worms, C. elegans with lin-12(n941)/glp-1 reduction-of-function did not show increasingly impaired octanol chemotaxis over time (p = 1.00).This is in contrast with wild-type (N2) and sel-12 mutant worms that demonstrated an increase in chemotaxis deficits over time (p<0.001).These findings suggest that Notch associated chemotaxis deficits do not fully phenocopy all of the characteristics of mutations in sel-12.

PLOS ONE
Presenilin-1 mediates amyloid-beta independent cell specific neurodegeneration in C. elegans sel-12 mutant C. elegans have ASH neuron morphological abnormalities that are rescued by wild-type PS1 and PS1 Δs169 , but not PS1 C410Y The chemotaxis experiments showed that sel-12 mutant worms had chemotaxis deficits that increased as worms aged.What causes the chemotaxis deficits?Since neuronal death is observed in AD we hypothesized that there might be neuronal degeneration occurring in sel-12 worms.This led us to investigate whether the chemotaxis behavioral phenotype reflected morphological changes or neurodegeneration of the sensory neurons.Inspired by results from past studies that showed that sel-12 loss-of-function mutants cause morphology defects in neurons corresponding to thermotaxis [51] and mechano-sensation [58] that aligned with observed behavioural phenotypes, we sought to study morphology of the ASH neurons what mediate our studied chemotaxis behaviour.To address whether the loss-of-function mutation in sel-12 altered the morphology of the ASH neurons, we crossed the sel-12(ok2078) mutant into a strain of worms expressing cytosolic GFP in the ASH neurons (osm-10::GFP), and using this resultant strain as the background generated transgenic lines over-expressing PS1 WT , PS1 Δs169 and PS1 C410Y .Using a confocal microscope we imaged the ASH neurons in separate groups of 100 wild-type, sel-12 mutant, Psra-6::PS1 WT , Psra-6::PS1 Δs169, and Psra-6::PS1 C410Y worms at 78, 98, and 108 hours of age.Observed neuron morphologies were categorized into "normal", "gapped", or "blebs" (Fig 6A).When we visualized the same GFP-filled ASH neurons using a DiI dye-filling protocol [59], the GFP co-localized with DiI dye, suggesting that the blebbing and gap phenotypes observed are indeed indicative of degenerated processes and not due to punctate or non-uniform distribution of GFP (Fig 6B).Within the same strain, there appeared to be no significant differences in the total number of abnormal ASH neurons across different ages (Fig 6C;p>0.05between all ages within the same strains).A chi-square test showed that sel-12 worms had significantly fewer normal ASH neurons compared to wildtype worms across all time points, X 2 (1, N = 200) = 16.33,p<0.01.However, as worms aged there appeared to be a progression from a "gapped arm" state to a more severe "blebbed" state in degenerating neurons-and this progression appeared more prominent in sel-12 mutants and PS1 C410Y over-expression animals.Animals with ASH neuron-specific expression of wildtype PS1 and PS1 Δs169 , had significantly more normal neurons than sel-12 animals and were not significantly different from wild-type, however animals with PS1 C410Y were not significantly different from sel-12 animals and had significantly fewer normal neurons than wildtype animals (Fig 6C).As a proxy measure for the health of the neurons the area of GFP in each neuron was quantified.ASH neurons in wild-type worms had significantly larger GFP area compared to sel-12 mutant worms at all time points; in sel-12 mutant animals the decrease in GFP area across age is particularly noticeable (Fig 6D).These data support the hypothesis that in sel-12 mutants the ASH neurons were degenerating as worms aged.This observed phenotype is rescued by human Wildtype PS1 and PS1 Δs169 over-expression but not PS1 C410Y.

Discussion
Our studies expand on previous evidence supporting the hypothesis of an Aβ-independent role of human PS1 in neural degeneration by studying sel-12, the C. elegans orthologue of PS1, in vivo.We demonstrated that in an Aβ-absent model, C410Y mutations in PS1 lead to ADlike pathogenesis and neuronal dysfunction in a cell-specific and cell-autonomous manner, whereas the Notch-intact Δs169 mutation largely rescues sel-12 mutant phenotypes.Importantly, expanding on previous studies that have alluded to the cell-autonomous function sel-12 [30,51,54], we demonstrate that the role of sel-12 in mediating chemotaxis acts in a cell-specific and cell-autonomous manner through double-dissociation (Figs 3 and 4 We observed that worms with sel-12 mutations had chemotaxis deficits from shortly after hatching, and that these deficits increased with age (Fig 1D).The ASH neurons that are responsible for detection of the octanol odorant showed morphological abnormalities indicative of neurodegeneration and degenerated more rapidly in sel-12 mutant worms compared to wild-type worms (Fig 6).This progressive neurodegeneration paralleled the decline of the chemotaxis indices of sel-12 mutant worms over time.Human PS1 and sel-12 have considerable conservation of function, such that the over-expression of human WT PS1 rescued sel-12 mutant phenotypes [30].Consistent with past findings, here we found that the over-expression of either wild-type sel-12 or human PS1 , in the ASH neurons rescued both chemotaxis deficits and ASH morphological abnormalities in sel-12 mutant worms (Figs 2 & 6).
Because sel-12 is strongly expressed in all C. elegans neurons, these data raise the question of whether the entire nervous system degenerates as a result of sel-12 mutations.Our behavioural data suggest that, in addition to the ASH neurons, the AWA sensory neurons may also be degenerating.Previous work on sel-12 worms examined the AIY interneurons and the mechanosensory neurons (ALM and PLM).Wittenburg et al. [51] showed that C. elegans with mutations in sel-12 have defects in thermotaxis behaviour as a result of a loss of function in cholinergic interneurons from the time of hatching [51].They also showed that these mutant worms displayed abnormal morphologies in the AIY interneurons, and that this phenotype was rescued by AIY-specific wild-type sel-12 and wild-type PS1 expression [51].Sarasija et al. [58] reported that mutations in sel-12 lead to metabolic defects of the mitochondria, resulting in oxidative stress and neurodegeneration in the ALM and PLM neurons responsible for responding to mechanical stimuli [58].Reducing calcium levels in the mitochondria in sel-12 mutant worms prevented neurodegeneration in mechanosensory neurons [58].Therefore, it is possible that mutations in sel-12 impact a number of neurons.Our locomotion data suggests that not all neurons are affected in mature sel-12 mutant worms as they were still able to move about on the agar surface, indicating that the motor neurons continue to drive movement even in the oldest worms we tested (S2A and S2B Fig) .C. elegans provides a unique system in which the role of presenilin in olfactory dysfunction and neuron death can be investigated using single cell rescues.As presenilins are substrates for different caspase proteins, the interaction between PS1 and caspases can be further explored.This use of single cell resolution phenotyping allows the characterization of even subtle and pleotropic phenotypes, providing potential novel therapeutic opportunities.
While findings from our experiments are consistent with other observations in the literature that demonstrate mechanistic loss-of-function phenotypes of PS1 function, our experiments do not reconcile this with the autosomal dominant nature of pathogenic PS mutations in fAD.In our experiments transgenes were over-expressed as extrachromosomal arrays leading to variability between the transgenic lines.We do not know the copy number of the transgenes expressed by these arrays (it could be a few copies or several hundred copies), and the expression level would likely have been much higher than what is found in vivo.This suggests to us that overexpressed levels of wildtype sel-12 and human PS1 do not cause any deficits in chemotaxis in C. elegans.Newer techniques such as the Mos10 mediated Single Copy Insertion (MosSCI) [60] and the CRISPR-Cas9 system can be used in C. elegans to generate single or low number copy rescues [61].Both approaches would allow a single copy of PS1 to replace mutant sel-12 in worms and might produce more consistent effects.Additionally, this approach can make testing phenotypes in a heterozygote possible and further resolve the paradox between the mechanistic loss-of-function hypothesis of PS and the autosomal dominant mode nature of pathogenic mutations of PS.Interestingly, in mice studies inactivation of a single PS1 locus is not sufficient to cause neurodegeneration while having one copy of a pathogenic variant is [62,63]-it is hypothesized, therefore, that the resulting mutant protein is antimorphic and in addition to being loss-of-function can act in a dominant-negative manner to inhibit the activities of normal PS produced from the remaining alleles to cause the dominant inheritance pattern of PS mutations.This hypothesis has yet to be fully explored.
This work together with previous studies [58,64] demonstrate that neurodegeneration of neurons in C. elegans with sel-12 mutations can occur in the absence of Aβ deposits.This offers an opportunity to study the role presenilins may play in neurodegeneration, independent of Aβ.While previous work suggested that the sel-12 neural degeneration was due to a Notchindependent mitochondrial dysfunction pathway [58], redundancy of function between glp-1 and lin-12 was not tested in these experiments.Our data suggests that although Notch contributes to the chemotaxis deficit phenotypes, double loss or knock-down of Notch receptors shows that the age-progressive phenomenon may be Notch-independent.
Perhaps the most interesting observation from our experiments was that whereas overexpression of human PS1 C410Y did not rescue sel-12 loss-of-function deficits, over-expression of the PS1 Δs169 variant rescued both the chemotaxis and neurodegeneration phenotypes.As PS1 Δs169 is hypothesized not to affect Notch signaling while PS1 C410Y does, and that PS1 Δs169 rescued both the chemotaxis and neurodegeneration phenotypes observed in our studies, it may be that Notch is an important signaling pathway for AD pathology.Indeed, the fact that decreased expression of both of the two Notch receptors lin-12 and glp-1 together exhibited chemotaxis deficits suggests that the phenotypes observed in this study are predominantly linked to Notch signalling [45].Indeed, Notch1 activation and expression has been shown to be significantly reduced in neurons in brain tissue and from AD patients [65] and altered Notch regulation can contribute to pathology of disease.Therefore, we investigated whether the interaction between PS1 and Notch played a role in these chemotaxis deficits by knocking down glp-1 with RNAi in a lin-12 mutant strain and confirmed the importance of Notch for the chemotaxis phenotype.Our data with PS1 Δs169 and the Notch knockdown experiments appears to suggest that in the C. elegans model the deficits caused by sel-12 loss-of-function and PS1 C410Y are derived largely from Notch signalling, which is contrary to the fact that the patient mutation PS1 Δs169 itself is a pathogenic variant.This could possibly be explained by C. elegans lacking a mammalian ortholog for APP and β-secretase that induce deleterious effects in mechanisms that are present in the mammalian system [29].However, we also found that the chemotaxis deficits induced by impairments in Notch signaling did not increase as animals aged (Fig 4).This suggests that although Notch plays an important role in octanol chemotaxis, the age-dependent and progressive component of the behavioural deficits we observed may be mediated by another Notch-independent pathway that sel-12/PS1 regulates.
To further examine whether the chemotaxis deficits and neurodegeneration we observed in our experiments are purely due to Notch or do indeed have a Notch-independent component, a more detailed analysis-including studying a sel-12;lin-12;glp-1 triple knockdown for both chemotaxis and neurodegeneration phenotypes to parse out an epistatic relationship between sel-12 and the Notch receptors-is required.However, the sel-12 strain and the lin-12/glp-1 strain used in this study are problematic to cross because hermaphrodites of both strains display the bag-of-worm phenotype due to improper development of vulval tissue [41,66] and males of both strains have significant mating deficiencies because of developmental defects in the male sex tissues [67,68].Additionally, the hsp::glp-1 RNAi construct is overexpressed as an extrachromosomal array and the mosaic inheritance enhances the difficulty in generating and selecting transgenic males across generations for a cross.As a result, we were not successful in generating a triple mutant as suggested above to further elaborate the involvement of Notch in our phenotypes.
It is also possible that loss of SEL-12/PS1 may cause some components of our observed phenotype through a Notch-independent and Amyloid-independent mechanism.Indeed, presenilin loss has been found to cause Ca2+ mediated mitochondrial dysfunction to drive neurodegeneration [54,58].Whether mitochondrial dysfunction, or another mechanism yet to be found, may mediate the phenotypes we observe in the sel-12 mutant and human PS1 over-expression strains would be an exciting direction to follow-up on.Studies interrogating other molecular pathways PS1 is involved in will uncover possible other pathways of PS1 that may contribute to fAD.
From the perspective of neurodegeneration the results we obtained can best be explained by two possibilities: one, that the fAD mutation has a lethal effect in neurons leading to cell death, or two, that presenilins play an important role in cell maintenance or survival and that classic fAD mutations interfere with that function and without it neurons die.Thus, a pathogenic mutation either kills the cell, or stops an essential function required for the cell to survive.This offers interesting research questions to pursue in the future.Our observation of cellspecific rescue shows that the effect of the mutation is cell specific and so is not a circuit issue, but an issue at the level of individual neurons.Loss-of-function of sel-12 has been linked to autophagy-mediated neurodegeneration in C. elegans [64], but more research is required to elucidate the roles other cell-death pathways may play in sel-12 mediated neurodegeneration as well.C. elegans offers the unique opportunity to study the functions of human disease genes and disease-causing mutations at the level of single neurons in living, behaving animals.This knowledge may lead to novel approaches to studying AD and developing new approaches to diagnosis and treatment of this debilitating disorder.

Fig 1 .
Fig 1. Worms with mutations in orthologs of PS1 show progressive chemosensory impairments.A) Chemotaxis assayworms are placed in the centre circle and allowed 1 hour to navigate plate.Anesthetic is included in buffer and odorant spots, so worms remain at the quadrant of choice.Worms in each quadrant are counted and CI calculated as shown.B) at 72 hours post egg-lay, young adult sel-12 mutants display impaired octanol avoidance behavior.C) sel-12 chemotaxis deficits are present from hatch.D) sel-12 chemotaxis deficits become more severe with age.At 78 hours post egg-lay worms are egg laying adults.They continue to lay eggs for 3-4 days.By 7 days post egg-lay worms are progressing into old age.Each bar represents an average from 4 independent plates (n = 50-100 worms per plate).Independent plate CI are shown as circles.White bars represent control plates and black bars represent test plates.Error bars reflect the standard error of the mean.*** p<0.001, *p<0.05.https://doi.org/10.1371/journal.pone.0289435.g001 [F(2,17) = 2.65, p<0.001]).Over-expressing human PS1 driven by the sel-12 promoter (Psel-12::PS1 WT ) also rescued the chemotaxis deficit.Chemotaxis tests were also conducted on wild-type worms, sel-12 mutant worms, and rescue lines over-expressing Psel-12:: PS1 WT in the sel-12 mutant background at 88, 98, and 108 hours of age (S3 Fig).For each age, Psel-12::PS1 WT rescue lines restored chemotaxis deficits in sel-12(2078) mutant worms and had CI values significantly different from sel-12 mutant worms (At 88 hours [F(2,17) = 67.98,

Fig 4 .Fig 5 .
Fig 4. Wildtype PS1 over-expression in the ASH neuron had a cell-specific effect on rescuing chemotaxis in sel-12 mutant background.Wildtype PS1 was expressed in AWA neurons by odr-10 promoter (responsible for diacetyl) and ASH neurons by sra-6 promoter (responsible for octanol) cell-specifically.A) Podr-10::PS1 did not rescue chemotaxis deficits in response to octanol.B) Podr-10::PS1 rescued chemotaxis deficits in response to diacetyl.C) Psra6::PS1 did not rescue chemotaxis deficits in response to diacetyl.Bars with a solid outline denote chemotaxis assays done with octanol, while dotted bars denote a chemotaxis assays done with diacetyl.Each bar represents an average from 4 independent plates (n = 50-100 synchronized 72 hr post egg-lay young adult worms per plate).Rescue bars are collapsed averages of 3 independent rescue lines generated.Independent plate CI are shown as circles.White bars represent control plates and black bars represent test plates.Error bars reflect the standard error of the mean.*** p<0.001.https://doi.org/10.1371/journal.pone.0289435.g004